IGF-1 receptor interacting proteins

ABSTRACT

The invention comprises a nucleic acid molecule with the sequence SEQ ID NO:5 and the complementary sequence, and its use in diagnosis and therapy. This nucleic acid molecule (IIP-10) is a gene which encodes an IGF-1 receptor binding polypeptide.

[0001] This is a divisional of copending application Ser. No.09/453,195, filed Dec. 2, 1999.

[0002] BACKGROUND OF THE INVENTION

[0003] The IGF-1 receptor signaling system plays an important role intumor proliferation and survival and is implicated in inhibition oftumor apoptosis. In addition and independent of its mitogenicproperties, IGF-1 R activation can protect against or at least retardprogrammed cell death in vitro and in vivo (Harrington et al., EMBO J.13 (1994) 3286-3295; Sell et al., Cancer Res. 55 (1995) 303-305;Singleton et al., Cancer Res. 56 (1996) 4522-4529). A decrease in thelevel of IGF-1R below wild type levels was also shown to cause massiveapoptosis of tumor cells in vivo (Resnicoff et al., Cancer Res. 55(1995) 2463-2469; Resnicoff et al., Cancer Res. 55 (1995) 3739-3741).Overexpression of either ligand (IGF) and/or the receptor is a featureof various tumor cell lines and can lead to tumor formation in animalmodels. Overexpression of human IGF-1R resulted in ligand-dependentanchorage-independent growth of NIH 3T3 or Rat-1 fibroblasts andinoculation of these cells caused a rapid tumor formation in nude mice(Kaleko et al., Mol. Cell. Biol. 10 (1990) 464-473; Prager et al., Proc.Natl. Acad. Sci. USA 91 (1994) 2181-2185). Transgenic miceoverexpressing IGF-II specifically in the mammary gland develop mammaryadenocarcinoma (Bates et al., Br. J. Cancer 72 (1995) 1189-1193) andtransgenic mice overexpressing IGF-II under the control of a moregeneral promoter develop an elevated number and wide spectrum of tumortypes (Rogler et al., J. Biol. Chem. 269 (1994) 13779-13784). Oneexample among many for human tumors overexpressing IGF-I or IGF-II atvery high frequency (>80%) are Small Cell Lung Carcinomas (Quinn et al.,J. Biol. Chem. 271 (1996) 11477-11483). Signaling by the IGF systemseems to be also required for the transforming activity of certainoncogenes. Fetal fibroblasts with a disruption of the IGF-1 R genecannot be transformed by the SV40 T antigen, activated Ha-ras, or acombination of both (Sell et al., Proc. Natl. Acad. Sci USA 90 (1993)11217-11221; Sell et al., Mol. Cell. Biol. 14 (1994) 3604-3612), and theE5 protein of the bovine papilloma virus is also no longer transforming(Morrione et al., J. Virol. 69 (1995) 5300-5303). Interference with theIGF/IGF-1R system was also shown to reverse the transformed phenotypeand to inhibit tumor growth (Trojan et al., Science 259 (1993) 94-97;Kalebic et al., Cancer Res. 54 (1994) 5531-5534; Prager et al., Proc.Natl. Acad, Sci. USA 91 (1994) 2181-2185; Resnicoffet al., Cancer Res.54 (1994) 2218-2222; Resnicoff et al., Cancer Res. 54 (1994) 4848-4850;Resnicoff et al., Cancer Res. 55 (1995) 2463-2469. For example, miceinjected with rat prostate adenocarcinoma cells (PA-III) transfectedwith IGF-1 R antisense cDNA (729 bp) develop tumors 90% smaller thancontrols or remained tumor-free after 60 days of observation (Burfeindet al., Proc. Natl. Acad. Sci. USA 93 (1996) 7263-7268). IGF-1R mediatedprotection against apoptosis is independent of de-novo gene expressionand protein synthesis. Thus, IGF-1 likely exerts its anti-apoptoticfunction via the activation of preformed cytosolic mediators.

[0004] Some signaling substrates which bind to the IGF-1R (e.g. IRS-1,SHC, p85 P13 kinase etc., for details see below) have been described.However, none of these transducers is unique to the IGF-1R and thuscould be exclusively responsible for the unique biological features ofthe IGF-1 R compared to other receptor tyrosine kinase including theinsulin receptor. This indicates that specific targets of the IGF-1 R(or at least the IGF-receptor subfamily) might exist which triggersurvival and counteract apoptosis and thus are prime pharmaceuticaltargets for anti-cancer therapy.

[0005] By using the yeast two-hybrid system it was shown that p85, theregulatory domain of phosphatidyl inositol 3 kinase (PI3K), interactswith the IGF-1R (Lamothe, B., et al., FEBS Lett. 373 (1995) 51-55;Tartare-Decker, S., et al., Endocrinology 137 (1996) 1019-1024). Howeverbinding of p85 to many other receptor tyrosine kinases of virtually allfamilies is also seen. Another binding partner of the IGF-1R defined bytwo-hybrid screening is SHC which binds also to other tyrosine kinasessuch as trk, met, EGF-R and the insulin receptor (Tartare-Deckert, S.,et al., J. Biol. Chem. 270 (1995) 23456-23460).

[0006] The insulin receptor substrate 1 (IRS-1) and insulin receptorsubstrate 2 (IRS-2) were also found to both interact with the IGF-1R aswell as the insulin receptor (Tartare-Deckert, S., et al., J. Biol.Chem. 270 (1995) 23456-23460; He, W., et al., J. Biol. Chem. 271 (1996)11641-11645; Dey, R. B., et al., Mol. Endocrinol. 10 (1996) 631-641).Grb 10 which interacts with the IGF-1R also shares many tyrosine kinasesas binding partners, e.g. met, insulin receptor, kit and abl (Dey, R.B., et al., Mol. Endocrinol. 10 (1996) 631-641; Morrione, A., et al.,Cancer Res. 56 (1996) 3165-3167). The phosphatase PTP1D (syp) shows alsoa very promiscuous binding capacity, i.e. binds to IGF-1R, insulinreceptor, met and others (Rocchi, S., et al., Endocrinology 137 (1996)4944-4952). More recently, mSH2-B and vav were described as binders ofthe IGF-1R, but interaction is also seen with other tyrosine kinases,e.g. mSH2-B also bind to ret and the insulin receptor (Wang, J., andRiedel, H., J. Biol. Chem. 273 (1998) 3136-3139). Taken together, the sofar described IGF-1 R binding proteins represent relatively unspecifictargets for therapeutic approaches, or are in the case of the insulinreceptor substrates (IRS-1, IRS-2) indispensable for insulin-drivenactivities.

SUMMARY OF THE INVENTION

[0007] The present invention relates to IGF-1 receptor interactingproteins (IIPs); nucleic acids coding therefor; and their use fordiagnostics and therapeutics, especially in the field of cancer. Inparticular, the invention relates to the identification of said genes inmammalian cells, especially in malignant tumor cells; to gene therapymethods for inhibiting the interaction between IGF-1 receptor and IIPs;methods of screening for potential cancer therapy agents; and cell linesand animal models useful in screening for and evaluating potentiallyuseful pharmaceutical agents that inhibit the interaction between IIiPsand IGF-1 receptor. The present invention relates in particular to thecloning and characterization of the gene IIP-11 and the gene productsthereof. Said gene products (polypeptides, mRNA) are especiallycharacterized as having the ability to modulate the IGF-1 receptorsignaling pathway. The function of the gene products according to theinvention is therefore to modulate signal transduction of the IGF-1receptor. Forced activation of IIPs therefore correlates with increasedtumor cell proliferation, survival and escape of apoptosis. It is anobject of the invention to provide novel genes encoding binding proteinsof IGF-1R as well as the corresponding polypeptides which modulate,preferably activate the IGF-1 receptor signaling pathway. It isenvisioned that this invention provides a basis for new cancer therapiesbased on the modulation, preferably inhibition, of the interactionbetween IGF-1R and IIPs.

DESCRIPTION OF THE FIGURES AND SEQUENCES

[0008]FIG. 1 Domain structure of yeast two-hybrid baits which were usedto screen cDNA libraries for cytoplasmic binding proteins of the IGF-1receptor. The LexA DNA binding domain was fused to the cytoplasmic (cp)domain (nt 2923 to 4154) of the wildtype IGF-1 receptor (a) or thekinase inactive mutant (K/A mutation at aa 1003) (b) (Ullrich, A., etal., EMBO J. 5 (1986) 2503-2512; Weidner, K. M., et al., Nature 384(1996) 173-176). The nucleotide and amino acid sequence of two differentlinkers inserted between the LexA DNA-binding domain and the receptordomain are shown below. The I1a(wt IGF-1 receptor) and K1 (kinaseinactive mutant IGF-1 receptor) constructs contain an additional prolineand glycine compared to the I2 and K2 constructs.

[0009]FIG. 2 Modification of the yeast two-hybrid LexA/IGF-1 receptorbait construct.

[0010] a) Schematic illustration of cytoplasmic binding sites of theIGF-1 receptor. The α-subunits of the IGF-1 receptor are linked to theβ-chains via disulfid bonds. The cytoplasmic part of the β-chaincontains binding sites for substrates in the juxtamembrane andC-terminal domain.

[0011] b) Domain structure of the two-hybrid bait containing only thejuxtamembrane IGF-1 receptor binding sites. The juxtamembrane domain ofthe IGF-1 receptor (nt 2923 to 3051) (Uflrich, A., et al., EMBO J. 5(1986) 2503-2512) was fused to the kinase domain of tprmet (nt 3456 to4229) (GenBank accession number: HSU19348).

[0012] c) Domain structure of the two-hybrid bait containing only theC-terminal IGF-1 receptor binding sites. The C-terminal domain of theIGF-1 receptor (nt 3823 to 4149) (Ullrich, A., et al., EMBO J. 5 (1986)2503-2512) was fused to the kinase domain of tprmet (nt 3456 to 4229)(GenBank accession number: HSU19348).

[0013]FIG. 3 Isoforms of IIP-1.

[0014] a) Delineation of the cDNA sequences of IIP-1 and IIP-1 (p26).Nucleotides are numbered above. The potential translation initiationsite within the IIP-1 cDNA is at position 63. The first ATG as potentialtranslation initiation site in the alternative splice variant IIP-1(p26) is at position 353. Both cDNAs contain a stop codon at position1062.

[0015] b) Domain structure of IIP-1 and IIP-1 (p26). Amino acidpositions are indicated above. In comparison to IIP-1 (p26) IIP-1contain additional 97 amino acids at the N-terminus. Both isoforms ofIIP-1 contain a PDZ domain spanning a region between amino acids 129 and213.

[0016]FIG. 4 Delineation of the IGF-1 receptor binding domain of IIP-1.Full-length IIP-1, its partial cDNA clones (IIP-1a and IIP-1b) anddeletion mutants (IIP-1a/mu1, IIP-1a/mu2, IIP-1a/mu3, IIP-1b/mu1) wereexamined for interaction with the IGF-1 receptor in the yeast two-hybridsystem. Yeast cells were cotransfected with a LexA IGF-1 receptor fusionconstruct and an activation plasmid coding for IIP-1 or the differentIIP-1 mutants fused to the VP16 activation domain. Interaction betweenIIP-1 or its mutants and the IGF-1 receptor was analyzed by monitoringgrowth of yeast transfectants plated out on histidine deficient mediumand incubated for 6d at 30° C. (diameter of yeast colonies: +++, >1 mmin 2d; ++, >1 mm in 4d; +, >1 mm in 6d; −, no detected growth). The PDZdomain can be defined as essential and sufficient for mediating theinteraction with the IGF-1 receptor. Nucleotide positions with respectto full length IIP-1 are indicated above.

[0017]FIG. 5 Protein sequence motifs of IIP-10. The amino acid sequenceof IIP-10 was analyzed using the computer program “Motifs” (WisconsinPackage Version 10.0, Genetics Computer Group (GCG), Madison, Wis.)which looks for protein motifs by searching protein sequences forregular expression patterns described in the PROSITE Dictionary.

[0018] SEQ ID NO:1 Nucleotide sequence of IIP-1 (cDNA).

[0019] SEQ ID NO:2 Predicted amino acid sequence of IIP-1.

[0020] SEQ ID NO:3 Nucleotide sequence of the IIP-6 partial cDNA clone.

[0021] SEQ ID NO:4 Deduced amino acid sequence of the IIP-6 partial cDNAclone. Cysteine and histidine residues of the two Cys₂His₂ Zinc fingerdomains are amino acids 72, 75, 88, 92, 100, 103, 116, and 120.

[0022] SEQ ID NO:5 Nucleotide sequence of IIP-10 (cDNA).

[0023] SEQ ID NO:6 Deduced amino acid sequence of IIP-10.

[0024] SEQ ID NO:7 Primer TIP2c-s.

[0025] SEQ ID NO:8 Primer TIP2b-r.

[0026] SEQ ID NO:9 Primer Hcthy-s.

[0027] SEQ ID NO:10 Primer Hcthy-r.

Detailed Description of the Invention

[0028] The present invention relates to IGF-1 receptor interactingproteins (IIPs); nucleic acids coding therefor; and their use fordiagnostics and therapeutics, especially in the field of cancer. Theinvention preferably comprises a nucleic acid encoding a protein(IIP-10) binding to IGF-1 receptor selected from the group comprising

[0029] a) the nucleic acids shown in SEQ ID NO:5 or a nucleic acidsequence which is complementary thereto,

[0030] b) nucleic acids which hybridize under stringent conditions withone of the nucleic acids from a) which encode a polypeptide showing atleast 75% homology with the polypeptide of SEQ ID NO:6 or

[0031] c) sequences that, due to the degeneracy of the genetic code,encode IIP-10 polypeptides having the amino acid sequence of thepolypeptides encoded by the sequences of a) and b).

[0032] The cDNA of IIP-10 codes for a new protein of 226 amino acid witha calculated molecular weight of 25.697. IIP-10 is a lysine rich protein(11%). IIP-10 contains an N-glycosylation site, several N-myristoylationsites, Ck2 and PKC phosphorylation sites, one tyrosine kinasephosphorylation site and one putative nuclear localization signal (FIG.5). The cDNA sequence of IIP-10 shows 65% homology to the cDNA sequenceof the Gallus Gallus thymocyte protein cthy28kD (EMBL accession number:GG34350). The amino acid sequences of IIP-10 and cthy28kD show 70%identity. Nt 383 to nt 584 of the IIP-10 cDNA are 94% identical to apartial cDNA described in WO 95/14772 (human gene signature HUMGS06271;accession number T24253). By immunofluorescence, flag-tagged IIP-10shows both a cytoplasmic and a nuclear localization in NIH3T3 cellsoverexpressing the IGF-1 receptor. Further yeast two-hybrid analysisrevealed that IIP-10 interacts in a phosphorylation dependent mannerwith the IGF-1 receptor. IIP-10 does not interact with the insulinreceptor. Deletion analysis of IIP-10 revealed that aa 19 to aa 226 aresufficient for binding to the IGF-1 receptor.

[0033] “Interaction” or “binding between IIP10 and the IGF-1 receptor”means a specific binding of the IIP10 polypeptide to the IGF-1 receptorbut not to control proteins such as lamin in the yeast two hybridsystem. Specific binding to the IGF-1 receptor can be demonstrated usingglutathion-S-transferase (GST)-IIP fusion proteins expressed in bacteriaand IGF-1 receptors expressed in mammalian cells. Furthermore, anassociation between a Flag tagged IIP-10 fusion protein (cf. Weidner, K.M. et al., Nature 384 (1996) 173-176) and the IGF-1 receptor can bemonitored in mammalian cell systems. For this purpose eukaryoticexpression vectors are used to transfect the respective cDNAs.Interaction between the proteins is visualized by coimmunoprecipitationexperiments or subcellular localization studies using anti-Flag oranti-IGF-1 receptor antibodies. Further provided by the invention areprobes and primers for the genes according to the invention, as well asnucleic acids which encode antigenic determinants of the gene productsaccording to the invention. Therefore, preferred embodiments includenucleic acids with preferably 10 to 50, or more preferably, 10 to 20consecutive nucleotides out of the disclosed sequences.

[0034] The term “nucleic acid” denotes a polynucleotide which can be,for example, a DNA, RNA, or derivatized active DNA or RNA. DNA and mRNAmolecules are preferred, however.

[0035] The term “hybridize under stringent conditions” means that twonucleic acid fragments are capable of hybridization to one another understandard hybridization conditions described in Sambrook et al.,Molecular Cloning: A laboratory manual (1989) Cold Spring HarborLaboratory Press, New York, USA.

[0036] More specifically, “stringent conditions” as used herein refersto hybridization in 5.0× SSC, 5× Denhardt, 7% SDS, 0.5 M phosphatebuffer pH 7.0, 10% dextran sulfate and 100 μg/ml salmon sperm DNA atabout 50° C.-68° C., followed by two washing steps with 1× SSC at 68° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperatures, about 22° C., to highstringency conditions at about 68° C.

[0037] The invention further comprises recombinant expression vectorswhich are suitable for the expression of IIP-10, recombinant host cellstransfected with such expression vectors, as well as a process for therecombinant production of a protein which is encoded by the IIP-10 gene.

[0038] The invention further comprises synthetic and recombinantpolypeptides which are encoded by the nucleic acids according to theinvention, and preferably encoded by the DNA sequence shown in SEQ IDNO:5 as well as peptidomimetics based thereon. Such peptidomimetics havea high affinity for cell membranes and are readily taken up by thecells. Peptidomimetics are preferably compounds derived from peptidesand proteins, and are obtained by structural modification usingunnatural amino acids, conformational restraints, isosterical placement,cyclization, etc. They are based preferably on 24 or fewer, preferably20 or fewer, amino acids, a basis of approximately 12 amino acids beingparticularly preferred.

[0039] The polypeptides and peptidomimetics can be defined by theircorresponding DNA sequences and by the amino acid sequences derivedtherefrom. The isolated IIP polypeptide can occur in natural allelicvariations which differ from individual to individual. Such variationsof the amino acids are usually amino acid substitutions. However, theymay also be deletions, insertions or additions of amino acids to thetotal sequence leading to biologically active fragments. The IIP proteinaccording to the invention—depending, both in respect of the extent andtype, on the cell and cell type in which it is expressed—can be inglycosylated or non-glycosylated form. IIP-Polypeptides with tumoricidicand/or metastatic activity can be identified by a tumor progressionassay using mammalian cells expressing said polypeptides and measuringthe proliferation capacity and apoptosis in relation to mammalian cellsnot expressing said polypeptides. “Polypeptide with IIP-10 activity orIIP-10” therefore means proteins with minor amino acid variations butwith substantially the same activity as IIP-10. Substantially the samemeans that the activities are of the same biological properties and thepolypeptides show at least 75% homology (identity) in amino acidsequences with IIP-10. More preferably, the amino acid sequences are atleast 90% identical. Homology according to the invention can bedetermined with the aid of the computer programs Gap or BestFit(University of Wisconsin; Needleman and Wunsch, J. Biol. Chem. 48 (1970)443-453; Smith and Waterman, Adv. Appl. Math. 2 (1981) 482-489). OtherIIPs according to, and used by, the invention are in particular:

[0040] IIP-1

[0041] A cDNA encoding an IGF-1 receptor interacting protein which wasnamed IIP-1 (SEQ ID NO: 1) was isolated. The cDNA of IIP-1 codes for anew protein of 333 aa with a calculated molecular weight of 35,727.IIP-1 is a glycine rich protein (13%). IIP-1 contains severalN-myristoylation sites, PKC and Ck2 phosphorylation sites and twoputative nuclear localization signals. A second isoform, IIP-I (p26), of236 aa in length with a calculated molecular weight of 26,071 wasidentified which was generated most likely by alternative splicing (FIG.3). Both isoforms bind to the IGF-1 receptor. cDNA sequences of IIP-Ihave been reported previously (Database EMBL Nos. AF089818 and AF061263;DeVries, L., et al., Proc. Natl. Acad. Sci. USA 95 (1998) 12340-12345).Two overlapping cDNA clones (FIG. 4) were identified which show highhomology to the human TIP-2 partial cDNA (GenBank accession number:AF028824) (Rousset, R., et al., Oncogene 16 (1998) 643-654) and weredesignated as IIP-1a and IIP-1b. The IIP-1a cDNA corresponds to nt 117to 751 of TIP-2. The IIP-1b cDNA shows besides TIP-2 sequences (nt 1 to106) additional 5′ sequences which are homologous to sequence Y2H35 ofWO 97/27296 (nt 25 to 158).

[0042] IIP-1a and IIP-1b both share the sequence coding for the PDZdomain of TIP-2 (nt 156 to 410) which is a known protein-proteininteraction domain (Ponting, C. P., et al., BioEssays 19 (1997)469-479). By deletion analysis the PDZ domain was determined as theessential and sufficient IGF-1 receptor binding domain of IIP-1 (FIG.4). Further yeast two-hybrid analysis revealed that binding of the IIP-1protein to the IGF-1 receptor is specific for this receptor tyrosinekinase. No interaction was seen to the insulin receptor or Ros. Receptortyrosine kinases of other families did not interact with IIP-1 (e.g.Met, Ret, Kit, Fms, Neu, EGF receptor). Thus, IIP-1 most likely is thefirst interaction protein shown to be specific for the IGF-1 receptortyrosine kinase. IIP-1 also binds to the kinase inactive mutant of theIGF-1 receptor.

[0043] IIP-2

[0044] IIP-2 was identified as a new binder of the cytoplasmic part ofthe IGF-1 receptor which corresponds to human APS (EMBL accessionnumber: HSAB520). APS has been previously isolated in a yeast two-hybridscreen using the oncogenic c-kit kinase domain as bait (Yokouchi, M., etal., Oncogene 15 (1998) 7-15). IIP-2 interacts with the IGF-1 receptorin a kinase dependent manner. Binding of IIP-2 was also observed toother members of the insulin receptor family (insulin receptor, Ros),but not to an unrelated receptor tyrosine kinase (Met). The region ofIIP-2 which was found to interact with the IGF-1 receptor corresponds tohuman APS (nt 1126 to 1674, EMBL Acc No. AB000520) contains the SH2domain of APS (nt 1249 to 1545).

[0045] IIP-3

[0046] IIP-3 was isolated as a new IGF-1 receptor interacting proteinand is identical to PSM (GenBank accession number: AF020526). PSM isknown as a PH and SH2 domain containing signal transduction proteinwhich binds to the activated insulin receptor (Riedel, H., et al., J.Biochem. 122 (1997) 1105-1113). A variant of PSM has also been described(Riedel, H., et al., J. Biochem. 122 (1997) 1105-1113). Binding of IIP-3to the IGF-1 receptor is dependent on tyrosyl phosphorylation of thereceptor. A cDNA clone corresponding to nt 1862 to 2184 of the variantform of PSM was identified. The isolated cDNA clone turned out to codefor the IGF-1 receptor binding region. The SH2 domain of PSM (nt 1864 to2148, EMBL Acc No. AF020526) is encoded by the sequence of the IIP-3partial cDNA clone isolated.

[0047] IIP-4

[0048] IIP-4 was isolated as a new interacting protein of thecytoplasmic domain of the IGF-1 receptor. IIP-4 corresponds to p59fyn, asrc-like tyrosine kinase (EMBL accession number: MMU70324 and human fynEM_HUM1:HS66H14) (Cooke, M. P., and Perlmutter, R. M., New Biol. 1(1989) 66-74). IIP-4 binds in a kinase dependent manner to the IGF-1receptor and to several other receptor tyrosine kinases as to theinsulin receptor and Met. The region of IIP-4 interacting with the IGF-1receptor (nt 665 to 1044) contains the SH2 domain of p59fyn (EMBL AccNo. U70324).

[0049] IIP-5

[0050] IIP-5 was isolated as a new IGF-1 receptor interacting protein.IIP-5 shows a high homology to the zinc finger protein Zfp38 (EMBLaccession number: MMZFPTA) and is at least 80% homologous to thecorresponding human gene. Zfp-38 is known as a transcription factor(Chowdhury, K., et al., Mech. Dev. 39 (1992) 129-142). IIP-5 interactsexclusively with the activated and phosphorylated IGF-1 receptor, butnot with a kinase inactive mutant. In addition to binding of IIP-5 tothe IGF-1 receptor interaction of IIP-5 with receptor tyrosine kinasesof the insulin receptor family (insulin receptor, Ros) was observed.IIP-5 does not bind to the more distantly related receptor tyrosinekinase Met.

[0051] One cDNA clone binding to the IGF-1 receptor which codes for nt756 to 1194 of Zfp38 (EMBL Acc No. MMZFPTA) and contains the first zincfinger (nt 1075 to 1158) was isolated. This domain is sufficient forbinding to the activated IGF-1 receptor.

[0052] IIP-6

[0053] IIP-6 was identified as a new IGF-1 receptor interacting protein.IIP-6 shows weak similarity to the zinc finger domain of Zfp29 (EMBLaccession number: MMZFP29). Zfp29 consists of a N-terminaltranscriptional activation domain and 14 C-terminal Cys²His² zincfingers (Denny, P., and Ashworth, A., Gene 106 (1991) 221-227). Bindingof IIP-6 to the IGF-1 receptor depends on phosphorylation of the IGF-1receptor kinase. IIP-6 also binds to the insulin receptor, but does notinteract with Met. The region of IIP-6 found to interact with the IGF-1receptor (SEQ ID NO:3, SEQ ID NO:4) contains two zinc finger domains ofthe Cys²His² type.

[0054] IIP-7

[0055] IIP-7 was isolated as a new IGF-1 receptor interacting proteinwhich corresponds to Pax-3 (EMBL accession number: MMPAX3R and humanPax3 EM-HUM2:S69369). Pax-3 is known as a DNA-binding protein beingexpressed during early embryogenesis (Goulding, M. D., et al., EMBO J.10 (1991) 1135-1147). IIP-7 binds in a phosphorylation dependent mannerto the IGF-1 receptor. IIP-7 also interacts with the insulin receptorand Met. A partial IIP-7 cDNA clone turned out to code for the IGF-1receptor binding domain of Pax3 (nt 815 to 1199, EMBL Acc No. MMPAX3R).This region contains the Pax-3 paired damain octapeptide (nt 853 to 876)and the paired-type homeodomain (nt 952 to 1134).

[0056] IIP-8

[0057] IIP-8 codes for the full-length cDNA of Grb7 (EMBL accessionnumber: MMGRB7P, human Grb7 EM_HUM1:AB008789). Grb7, a PH domain and aSH3 domain containg signal transduction protein, was first published asan EGF receptor-binding protein (Margolis, B. L., et al., Proc. Natl.Acad. Sci. USA 89 (1992) 8894-8898). IIP-8 does not interact with thekinase inactive mutant of the IGF-1 receptor. Binding of IIP-8 toseveral other receptor tyrosine kinases (e.g. insulin receptor, Ros andMet) was also observed.

[0058] IIP-9

[0059] IIP-9 was identified as a new IGF-1 receptor interaction protein.IIP-9 is identical to nck-beta (EMBL Acc No. AF043260). Nck is acytoplasmic signal transduction protein consisting of SH2 and SH3domains (Lehmann, J. M., et al., Nucleic Acids Res. 18 (1990) 1048).IIP-9 interacts with the IGF-1 receptor in a phosphorylation dependentmanner. nck binds to the juxtamembrane region of the IGF-1 receptor.Apart from binding of IIP-9 to the IGF-1 receptor, interaction with theinsulin receptor but not with Ros or Met was seen.

[0060] A preferred object of the invention are polypeptides that arehomologous, and more preferably, polypeptides that are substantiallyidentical to the polypeptides of SEQ ID NO:6 (IIP-10). Homology can beexamined by using the FastA algorithm described by Pearson, W. R.,Methods in Enzymology 183 (1990) 63-68, Academic Press, San Diego, US.By “substantially identical” is meant an amino acid sequence whichdiffers only by conservative amino acid substitutions, for examplesubstitutions of one amino acid for another of the same class (e.g.valine for glycine, arginine for lysine, etc.) or by one or morenon-conservative amino acid substitution, deletions or insertionslocated at positions of the amino acid sequence which do not destroy thebiological function of the polypeptide. This includes substitution ofalternative covalent peptide bonds in the polypeptide. By “polypeptide”is meant any chain of amino acids regardless of length orposttranslational modification (e.g., glycosylation or phosphorylation)and can be used interchangeably with the term “protein”.

[0061] According to the invention by “biologically active fragment” ismeant a fragment which can exert a physiological effect of thefull-length naturally-occurring protein (e.g., binding to its biologicalsubstrate, causing an antigenic response, etc.).

[0062] The invention also features fragments of the polypeptideaccording to the invention which are antigenic. The term “antigenic” asused herein refers to fragments of the protein which can induce aspecific immunogenic response, e.g. an immunogenic response which yieldsantibodies which specifically bind to the protein according to theinvention. The fragments are preferably at least 8 amino acids, andpreferably up to 25 amino acids, in length. In one preferred embodiment,the fragments include the domain which is responsible for the binding ofthe IIPs to the IGF-1 receptor (i.e., the PDZ domain of IIP-1. By“domain” is meant the region of amino acids in a protein directlyinvolved in the interaction with its binding partner. PDZ domains areapproximately 90-residue repeats found in a number of proteinsimplicated in ion-, channel and receptor clustering and the linking ofreceptors to effector enzymes. Such PDZ are described in general byCabral, J. H., et al., Nature 382 (1996) 649-652.

[0063] The invention further comprises a method for producing a proteinaccording to the invention whose expression or activation is correlatedwith tumor proliferation, by expressing an exogenous DNA in prokaryoticor eukaryotic host cells and isolation of the desired protein orexpressing said exogeneous DNA in vivo for pharmaceutical means, whereinthe protein is coded preferably by a DNA sequence coding for IIP-10,preferably the DNA sequence shown in SEQ ID NO:5.

[0064] The polypeptides according to the invention can also be producedby recombinant means, or synthetically. Non-glycosylated IIP-10polypeptide is obtained when it is produced recombinantly inprokaryotes. With the aid of the nucleic acid sequences provided by theinvention it is possible to search for the IIP-10 gene or its variantsin genomes of any desired cells (e.g. apart from human cells, also incells of other mammals), to identify these and to isolate the desiredgene coding for the IIP-10 protein. Such processes and suitablehybridization conditions (see also above, “stringent conditions”) areknown to a person skilled in the art and are described, for example, bySambrook et al., Molecular Cloning: A laboratory manual (1989) ColdSpring Harbor Laboratory Press, New York, USA, and Hames, B. D.,Higgins, S. G., Nucleic acid hybridisation—apractical approach (1985)IRL Press, Oxford, England. In this case the standard protocolsdescribed in these publications are usually used for the experiments.

[0065] The use of recombinant DNA technology enables the production ofnumerous active IIP-10 derivatives. Such derivatives can, for example,be modified in individual or several amino acids by substitution,deletion or addition. The derivatization can, for example, be carriedout by means of site directed mutagenesis. Such variations can be easilycarried out by a person skilled in the art (J. Sambrook, B. D. Hames,loc. cit.). It merely has to be ensured by means of the below-mentionedtumor cell growth inhibition assay that the characteristic properties ofIIP-10 are preserved.

[0066] With the aid of such nucleic acids coding for an IIP-10 protein,the protein according to the invention can be obtained in a reproduciblemanner and in large amounts. For expression in prokaryotic or eukaryoticorganisms, such as prokaryotic host cells or eukaryotic host cells, thenucleic acid is integrated into suitable expression vectors, accordingto methods familiar to a person skilled in the art. Such an expressionvector preferably contains a regulatable/inducible promoter. Theserecombinant vectors are then introduced for the expression into suitablehost cells such as, e.g., E. coli as a prokaryotic host cell orSaccharomyces cerevisiae, teratocarcinoma cell line PA-1 sc 9117(Büttner et al., Mol. Cell. Biol. 11 (1991) 3573-3583), insect cells,CHO or COS cells as eukaryotic host cells and the transformed ortransduced host cells are cultured under conditions which allowexpression of the heterologous gene. The isolation of the protein can becarried out according to known methods from the host cell or from theculture supernatant of the host cell. Such methods are described forexample by Ausubel I., Frederick M., Current Protocols in Mol. Biol.(1992), John Wiley and Sons, New York. Also in vitro reactivation of theprotein may be necessary if it is not found in soluble form in the cellculture.

[0067] The invention therefore in addition concerns a IIP polypeptidewhich is a product of prokaryotic or eukaryotic expression of anexogenous DNA.

[0068] The protein can be isolated from the cells or the culturesupernatant and purified by chromatographic means, preferably by ionexchange chromatography, affinity chromatography and/or reverse phaseHPLC.

[0069] IIP-10 can be purified after recombinant production by affinitychromatography using known protein purification techniques, includingimmunoprecipitation, gel filtration, ion exchange chromatography,chromatofocussing, isoelectric focussing, selective precipitation,electrophoresis, or the like.

[0070] Diagnostic Methods:

[0071] The invention further comprises a method for detecting a nucleicacid molecule encoding an IIP-gene, comprising incubating a sample(e.g., body fluids such as blood, cell lysates) with a nucleic acidmolecule according to the invention and determining hybridization understringent conditions of said nucleic acid molecule to a target nucleicacid molecule for determination of presence of a nucleic acid moleculewhich is said IIP gene and therefore a method for the identification ofIGF-1R activation or inhibition in mammalian cells or body fluids.

[0072] Therefore the invention also includes a method for the detectionof the proliferation potential of a tumor cell comprising

[0073] a) incubating a sample of body fluid of a patient suffering fromcancer, a sample of cancer cells, or a sample of a cell extract or acell culture supernatant of said cancer cells, whereby said samplecontains nucleic acids with a nucleic acid probe which is selected fromthe group consisting of

[0074] (i) the nucleic acids shown in SEQ ID NOS:1, 3 or 5 or a nucleicacid which is complementary thereto and

[0075] (ii) nucleic acids which hybridize under stringent conditionswith one of the nucleic acids from (i) and

[0076] b) detecting hybridization by means of a further binding partnerof the nucleic acid of the sample and/or the nucleic acid probe or byX-ray radiography.

[0077] Hybridization between the probe and nucleic acids from the sampleindicates the presence of the RNA of such proteins. Such methods areknown to a person skilled in the art and are described, for example, inWO 89/06698, EP-A 0 200 362, U.S. Pat. No. 2,915,082, EP-A 0 063 879,EP-A 0 173 251, EP-A 0 128 018.

[0078] In a preferred embodiment of the invention the coding nucleicacid of the sample is amplified before the test, for example by means ofthe known PCR technique. Usually a derivatized (labeled) nucleic acidprobe is used within the framework of nucleic acid diagnostics. Thisprobe is contacted with a denatured DNA or RNA from the sample which isbound to a carrier and in this process the temperature, ionic strength,pH and other buffer conditions are selected—depending on the length andcomposition of the nucleic acid probe and the resulting meltingtemperature of the expected hybrid—such that the labeled DNA or RNA canbind to homologous DNA or RNA (hybridization see also Wahl, G. M., etal., Proc. Natl. Acad. Sci. USA 76 (1979) 3683-3687). Suitable carriersare membranes or carrier materials based on nitrocellulose (e.g.,Schleicher and Schüll, BA 85, Amersham Hybond, C.), strengthened orbound nitrocellulose in powder form or nylon membranes derivatized withvarious functional groups (e.g., nitro groups) (e.g., Schleicher andSchüll, Nytran; NEN, Gene Screen; Amersham Hybond M.; Pall Biodyne).

[0079] Hybridizing DNA or RNA is then detected by incubating the carrierwith an antibody or antibody fragment after thorough washing andsaturation to prevent unspecific binding. The antibody or the antibodyfragment is directed towards the substance incorporated duringderivatization into the nucleic acid probe. The antibody is in turnlabeled. However, it is also possible to use a directly labeled DNA.After incubation with the antibodies it is washed again in order to onlydetect specifically bound antibody conjugates. The determination is thencarried out according to known methods by means of the label on theantibody or the antibody fragment.

[0080] The detection of the expression can be carried out for exampleas:

[0081] in situ hybridization with fixed whole cells, with fixed tissuesmears and isolated metaphase chromosomes,

[0082] colony hybridization (cells) and plaque hybridization (phages andviruses),

[0083] Southern hybridization (DNA detection),

[0084] Northern hybridization (RNA detection),

[0085] serum analysis (e.g., cell type analysis of cells in the serum byslot-blot analysis),

[0086] after amplification (e.g., PCR technique).

[0087] Preferably the nucleic acid probe is incubated with the nucleicacid of the sample and the hybridization is detected optionally by meansof a further binding partner for the nucleic acid of the sample and/orthe nucleic acid probe.

[0088] The nucleic acids according to the invention are hence valuableprognostic markers in the diagnosis of the metastatic and progressionpotential of tumor cells of a patient.

[0089] Screening for Antagonists and Agonists of IIPs or Inhibitors

[0090] According to the invention antagonists of IIP-10 or inhibitorsfor the expression of IIP (e.g., antisense nucleic acids) can be used toinhibit tumor progression and cause massive apoptosis of tumor cells invivo, preferably by somatic gene therapy. Therefore, the presentinvention also relates to methods of screening for potentialtherapeutics for cancer, diabetes, neurodegenerative disorders, bonediseases, to methods of treatment for disease and to cell lines andanimal models useful in screening for and evaluating potentially usefultherapies for such disease. Therefore another object of the inventionare methods for identifying compounds which have utility in thetreatment of the afore-mentioned and related disorders. These methodsinclude methods for modulating the expression of the polypeptidesaccording to the invention, methods for identifying compounds which canselectively bind to the proteins according to the invention and methodsof identifying compounds which can modulate the activity of saidpolypeptides. These methods may be conducted in vitro and in vivo andmay employ the transformed cell lines and transgenic animal models ofthe invention.

[0091] An antagonist of IIPs or an inhibitor of IIP is defined as asubstance or compound which inhibits the interaction between IGF-1R andIIP, preferably IIP-10. Therefore the biological activity of IGF-1Rdecreases in the presence of such a compound. In general, screeningprocedures for IIP antagonists involve contacting candidate substanceswith IIP-bearing host cells under conditions favorable for binding andmeasuring the extent of decreasing receptor mediated signaling (in thecase of an antagonist). Such an antagonist is useful as a pharmaceuticalagent for use in tumor therapy. For the treatment of diabetes, neuraldiseases, or bone diseases, stimulation of the signaling pathway isrequired, i.e., screening for agonists is useful.

[0092] IIP activation may be measured in several ways. Typically, theactivation is apparent by a change in cell physiology such as anincrease or decrease in growth rate or by a change in thedifferentiation state or by a change in cell metabolism which can bedetected in standard cell assays, for example MTT or XTT assays (RocheDiagnostics GmbH, DE).

[0093] The nucleic acids and proteins according to the invention couldtherefore also be used to identify and design drugs which interfere withthe interaction of IGF-1R and IIPs. For instance, a drug that interactswith one of the proteins could preferentially bind it instead ofallowing binding its natural counterpart. Any drug which could bind tothe IGF-1 receptor and, thereby, prevent binding of an IIP or, viceversa, bind to an IIP and, thereby, prevent binding of the IGF-1receptor. In both cases, signal transduction of the IGF-1 receptorsystem would be modulated (preferably inhibited). Screening drugs forthis facility occurs by establishing a competitive assay (assay standardin the art) between the test compound and interaction of IIP and theIGF-1 receptor and using purified protein or fragments with the sameproperties as the binding partners.

[0094] The protein according to the invention is suitable for use in anassay procedure for the identification of compounds which modulate theactivity of the proteins according to the invention. Modulating theactivity as described herein includes the inhibition or activation ofthe protein and includes directly or indirectly affecting the normalregulation of said protein activity. Compounds which modulate theprotein activity include agonists, antagonists and compounds whichdirectly or indirectly affect the regulation of the activity of theprotein according to the invention. The protein according to theinvention may be obtained from both native and recombinant sources foruse in an assay procedure to identify modulators. In general, an assayprocedure to identify modulators will contain the IGF receptor, aprotein of the present invention, and a test compound or sample whichcontains a putative modulator of said protein activity. The testcompounds or samples may be tested directly on, for example, purifiedprotein of the invention, whether native or recombinant, subcellularfractions of cells producing said protein, whether native orrecombinant, and/or whole cells expressing said protein, whether nativeor recombinant. The test compound or sample may be added to the proteinaccording to the invention in the presence or absence of knownmodulators of said protein. The modulating activity of the test compoundor sample may be determined by, for example, analyzing the ability ofthe test compound or sample to bind to said protein, activate saidprotein, inhibit its activity, inhibit or enhance the binding of othercompounds to said protein, modifying receptor regulation or modifyingintracellular activity.

[0095] The identification of modulators of the protein activity areuseful in treating disease states involving the protein activity. Othercompounds may be useful for stimulating or inhibiting the activity ofthe protein according to the invention. Such compounds could be of usein the treatment of diseases in which activation or inactivation of theprotein according to the invention results in either cellularproliferation, cell death, non-proliferation, induction of cellularneoplastic transformations, or metastatic tumor growth and hence couldbe used in the prevention and/or treatment of cancers such as, forexample, prostate and breast cancer. The isolation and purification of aDNA molecule encoding the protein according to the invention would beuseful for establishing the tissue distribution of said protein as wellas establishing a process for identifying compounds which modulate theactivity of said protein and/or its expression.

[0096] Therefore a further embodiment of the invention is a method forscreening a compound that inhibits the interaction between IGF-1R andIIP-1, IIP-2, IIP3, IIP4, IIP5, IIP6, IIP7, IIP8, IIP9 or IIP-10,comprising

[0097] a) combining IGF-1R and the IIP polypeptide with a solutioncontaining a candidate compound such that the IGF-1 R and the IIPpolypeptide are capable of forming a complex and

[0098] b) determining the amount of complex relative to thepredetermined level of binding in the absence of said candidate compoundand therefrom evaluating the ability of said candidate compound toinhibit binding of IGF-1R to the IIP polypeptide.

[0099] Such a screening assay is preferably performed as an ELISA assaywhereby IGF-1R or the IIP-polypeptide preferably IIP-1 or IIP-10, isbound on a solid phase. A further embodiment of the invention is amethod for the production of a therapeutic agent for the treatment ofcarcinomas in a patient comprising combining a therapeutically effectiveamount of a compound which inhibits the interaction between IGF-1R andIIP in biochemical and/or cellular assays to an extent of at least 50%.Biochemical assays are preferably ELISA-based assays or homogeneousassays. In the case of the ELISA system antibodies specific for the twobinding partners are used for detection of the complexes. In the case ofthe homogenous assay at least one binding partner is labeled withfluorophores which allows analysis of the complexes. Cellular assays arepreferably assays whereby tumor cells or cells transfected withexpression constructs of the IGF-1 R and the respective binding proteinsare treated with or without drugs and complex formation between the twocomponents is then analyzed using standard cell assays.

[0100] A preferred embodiment of the invention is a method for theproduction of a therapeutic agent for the treatment of carcinomas in apatient comprising combining a pharmaceutically acceptable carrier witha therapeutically effective amount of a compound which inhibits theinteraction between IGF-1R and an IIP-polypeptide, preferably IIP-1 orIIP-10, in a cellular assay, whereby in said cellular assay tumor cellsor cells transfected with expression constructs of IGF-1 R and of therespective IIP are treated with said compound, and complex formationbetween IGF-1R and said respective IIP is analyzed, and the extent ofsaid complex formation in the case of inhibition does not exceed 50%referred to 100% for complex formation without said compound in saidsame cellular assay.

[0101] A further embodiment of the invention is a method of treating apatient suffering from a carcinoma with a therapeutically effectiveamount of a compound which inhibits the interaction between IGF-1R andthe IIP-polypeptide, preferably IIP-1 or IIP-10, in a cellular assay,whereby in said cellular assay tumor cells or cells transfected withexpression constructs of IGF-1 R and of the respective IIP are treatedwith said compound, and complex formation between IGF-1R and saidrespective IIP is analyzed, and the extent of said complex formation inthe case of inhibition does not exceed 50% referred to 100% for complexformation without said compound in said same cellular assay.

[0102] A further embodiment of the invention is an antibody againstIIP-1 or IIP-10 according to the invention.

[0103] Antibodies were generated from the human, mouse, or ratpolypeptides. Antibodies specifically recognizing IIP-1 or IIP-10 areencompassed by the invention. Such antibodies are raised using standardimmunological techniques. Antibodies may be polyclonal or monoclonal ormay be produced recombinantly such as for a humanized antibody. Anantibody fragment which retains the ability to interact with IIP-1 orIIP-10 is also provided. Such a fragment can be produced by proteolyticcleavage of a full-length antibody or produced by recombinant DNAprocedures. Antibodies of the invention are useful in diagnostic andtherapeutic applications. They are used to detect and quantitate IIP-1or IIP-10 in biological samples, particularly tissue samples and bodyfluids. They are also used to modulate the activity of IIP-1 or IIP-10by acting as an agonist or an antagonist.

[0104] The following examples, references, sequence listing and drawingare provided to aid the understanding of the present invention, the truescope of which is set forth in the appended claims. It is understoodthat modifications can be made in the procedures set forth withoutdeparting from the spirit of the invention.

EXAMPLES Example 1

[0105] Isolation and Characterization of IGF-1R Binding Proteins

[0106] The yeast two-hybrid system (Fields, S., and Song, O., Nature 340(1989) 245-246) was used to isolate unknown cytosolic IGF-1 receptorbinding proteins. For screening a modified version of the yeasttwo-hybrid system was used which allows interchaintyrosylphosphorylation of the receptors in yeast. The yeast two-hybridbait plasmid (BTM116-cpIGF-1 receptor) was constructed by fusing thecytoplasmic domain of the β-subunit of the IGF-1 receptor (nt 2923 to4154) (Ullrich, A., et al., EMBO J. 5 (1986) 2503-2512) to the LexADNA-binding domain which forms dimers and mimics the situation of theactivated wildtype receptor (cf. Weidner, M., et al., Nature 384 (1996)173-176). By introducing a proline-glycine spacer between the LexADNA-binding domain and the receptor domain the ability of the bait tobind known substrates of the IGF-1 receptor was remarkably increased incomparision to other spacer amino acids (FIG. 1).

[0107] Alternatively a bait was constructed containing only thejuxtamembrane or C-terminal region of the IGF-1 receptor (nt 2923 to3051 or nt 3823 to 4146) (Ullrich, A., et al., EMBO J. 5 (1986)2503-2512) fused to the kinase domain of an unrelated, very potentialreceptor tyrosine kinase. Here the kinase domain of tpr met (nt 3456 to4229) (GenBank accession number: HSU19348) (FIG. 2) was used. In thisway it is possible to delineate the region of the IGF-1 receptor whichmediates binding to downstream effectors. The IGF-1 receptor baitplasmid was used to screen activation domain cDNA libraries (e.g. VP16-or Gal4 based activation domain) (cf. Weidner, M., et al., Nature 384(1996) 173-176). The bait and prey plasmids were co-transfected intoSaccharomyces cerevisiae strain L40 containing a HIS3 and lacZ reportergene. Library plasmids were isolated from yeast colonies growing onhistidine deficient medium, were sequenced and reintroduced into yeaststrain L40. By co-transfecting experiments with different test baits,i.e. BTM 116 plasmids coding for a kinase inactive mutant of the IGF-1receptor (L1033A) or the cytoplasmic domain of receptor tyrosine kinasesof the insulin receptor family (insulin receptor, Ros) and of unrelatedreceptor tyrosine kinase families (Met, EGF receptor, Kit, Fms, Neu) thespecificity of the putative bait-prey interactions was evaluated.Several cDNAs were identified which code for previously unknown IGF-1receptor interacting proteins (IIPs). In addition binding domains ofknown substrates of the IGF-1 receptor such as the C-terminal SH2 domainof p85PI3K and the SH2 domain of Grb10 were found. The results are shownin Table 1. TABLE 1 IIP wt IGF-1R mu IGF-1R IR Ros Met IIP-1 + + − − −IIP-2 + − + + − IIP-3 + − + + + IIP-4 + − + nd + IIP-5 + − + + − IIP-6 +− + nd − IIP-7 + − + nd + IIP-8 + − + + + IIP-9 + − + − − IIP-10 + − −nd nd

[0108] Delineation of the binding specificity of the IIPs with respectto different receptor tyrosine kinases tested in the yeast two-hybridsystem. Yeast cells were cotransfected with a LexA fusion constructcoding for the different receptor tyrosine kinases and an activationplasmid coding for the different IIPs fused to the VP16 activationdomain. Interaction between the IIPs and the different receptor tyrosinekinases was analyzed by monitoring growth of yeast transfectants platedout on histidine deficient medium and incubated for 3d at 30° C. (wtIGF-1R, kinase active IGF-1 receptor; mu IGF-1 R, kinase inactive mutantIGF-1 receptor; IR, insulin receptor; Ros, Ros receptor tyrosine kinase;Met, Met receptor tyrosine kinase; +, growth of yeast transfectantswithin 3 days larger than 1 mm in diameter; −, no detected growth; nd,not determined).

Example 2

[0109] Assay Systems:

[0110] A) In-vitro/biochemical Assays:

[0111] ELISA-based assay/homogenous assay

[0112] IGF-1R and the binding proteins (IIPs) are expressed with orwithout Tag-epitopes in E.coli or eucaryotic cells and purified tohomogeneity. Interaction of IGF-1R and the respective binding proteinsare analyzed in the presence or absence of drugs. Compounds which eitherinhibit or promote binding of IGF-1R and the respective binding proteinsare selected. In the case of the ELISA system antibodies specific forthe two binding partners are used for detection of the complexes. In thecase of the homogenous assay at least one binding partner is labeledwith fluorophores which allows analysis of the complexes. Alternatively,anti-Tag-antibodies are used to monitor interaction.

[0113] B) Cellular Assays:

[0114] Tumor cells or cells transfected with expression constructs ofthe IGF-1 R and the respective binding proteins are treated with orwithout drugs and complex formation between the two components is thenanalyzed using standard assays.

Example 3

[0115] cDNA cloning of IIP-1 and IIP-10 (and RT-PCR-assay)

[0116] The nucleotide sequence of full length IIP-1 was determined bysequencing of the partial cDNA clones of IIP-1 and (IIP-1a, IIP-1b) andby using database information (ESTs). cDNA cloning of full length IIP-1was performed by RT PCR on total RNA isolated from a MCF7_(ADR)breastcell line. PT PCR with two oligonucleotide primers: TIP2c-s (SEQ IDNO:7) and TIP2b-r (SEQ ID NO:8) resulted in amplification of two DNAfragments of 1.0 kb (IIP-1) and 0.7 kb (IIP-1(p26)).

[0117] The nucleotide sequence of full length IIP-10 was determined bysequencing of the partial cDNA clone of IIP-10 and by using databaseinformation (ESTs). cDNA cloning of IIP-10 was performed on total RNAisolated from the colon cancer cell line SW480. RT PCR with twooligonucleotide primers: Hethy-s (SEQ ID NO:9) and Hcthy-r (SEQ ID NO:10) resulted in amplification of a cDNA fragment of 676bp (IIP-10). DNAsequencing was performed using the dideoxynucleotide chain terminationmethod on an ABI 373A sequencer using the Ampli Taq® FSDideoxyterminator kit (Perkin Elmer, Foster City, Calif.). Comparison ofthe cDNA and deduced protein sequences was performed using AdvancedBlast Search (Altschul, S. F., et al., J. Mol. Biol. 215 (1990) 403-410;Altschul, S. F., et al., Nucleic Acids Res. 25 (1997) 3389-3402).

Example 4

[0118] Western Blot Analysis of IIP-1 and IIP-10

[0119] Total cell lysates were prepared in a buffer containing 50 mMTris pH 8.0, 150 mM NaCl, 1% NP40, 0.5% deoxycholic acid, 0.1% SDS, and1 mM EDTA and cleared by centrifugation for 15 min at 4° C. The proteinconcentration of the supernatants was measured using the Micro BCAProtein Assay kit (Pierce Chemical Co., Rockford, Ill.) according to themanufacturer's manual. IGF-1 receptors were immunoprecipitated usinganti-IGF-1 receptor antibodies (Santa Cruz). Proteins were fractionatedby SDS-PAGE and electrophoretically transferred to nitrocellulosefilters. Nitrocellulose filters were preincubated with 10% (w/v)fat-free milk powder in 20 mM Tris pH 7.5, 150 mM NaCl, 0.2% Tween-20.Binding of a mouse monoclonal antibody directed against the flag epitopewas detected by horseradish peroxidase-labeled goat-anti-mouse IgGantiserum (Biorad, Munich, DE) and visualized using an enhancedchemoluminescence detection system, ECL™ (Amersham, Braunschweig, DE).

Example 5

[0120] Overexpression of IIP-1 to IIP-10 in Mammalian Cells byLiposome-mediated Transfection

[0121] The cDNAs for IIP-1 to -10 were cloned into the NotI site ofpBATflag or pcDNA3flag (Weidner, K. M., et al., Nature 384 (1996)173-176); Behrens, J., et al., Nature 382 (1996) 638-642; Behrens, J.,et al., Science 280 (1998) 596-599). NIH3T3 cells or other recipientcells were transfected with pcDNAflagIIP-1 to -10 or alternatively withpBATflag IIP-1 to -10 using FuGENE6 (Roche Biochemicals) as transfectionagent. Cells were selected in 0.4 mg/ml G418. Single clones were pickedand analzyed for expression of IIP-1 to -10 and functionallycharacterized with respect to proliferation.

[0122] Northern Blot Analysis

[0123] Human and murine mRNA multiple tissue Northern blots werepurchased from Clontech (Palo Alto, Calif., US). A cDNA probe spanningIIP-10 nt343-nt676 of the coding region was labeled with DIG-dUTP usingthe PCR DIG Labeling Mix (Roche Diagnostics GmbH, DE). A digoxygeninlabeled actin RNA probe was purchased from Roche Diagnostics GmbH, DE.Hybridization was performed using the DIG EasyHyb hybridization solution(Roche Diagnostics GmbH, DE). IIP-10 mRNA was detected with DIG-specificantibodies conjugated to alkaline phosphatase and the CSPD substrate(Roche Diagnostics GmbH, DE).

Example 6

[0124] Detection of mRNA in Cancer Cells

[0125] In order to detect whether proteins are expressed in cancer cellswhich are coded by nucleic acids which hybridize with SEQ ID NO:1 or SEQID NO:5 or the complementary sequence and consequently whether MRNA ispresent, it is possible on the one hand to carry out the establishedmethods of nucleic acid hybridization such as Northern hybridization,in-situ hybridization, dot or slot hybridization and diagnostictechniques derived therefrom (Sambrook et al., Molecular Cloning: Alaboratory manual (1989) Cold Spring Harbor Laboratory Press, New York,USA; Hames, B. D., Higgins, S. G., Nucleic acid hybridisation—apractical approach (1985) IRL Press, Oxford, England; WO 89/06698; EP-A0 200 362; EP-A 0 063 879; EP-A 0 173 251; EP-A 0 128 018). On the otherhand it is possible to use methods from the diverse repertoire ofamplification techniques using specific primers (PCR Protocols—A Guideto Methods and Applications (1990), publ. M. A. Innis, D. H. Gelfand, J.J. Sninsky, T. J. White, Academic Press Inc.; PCR—A Practical Approach(1991), publ. M. J. McPherson, P. Quirke, G. R. Taylor, IRL Press).

[0126] The RNA for this is isolated from the cancer tissue by the methodof Chomcszynski and Sacchi, Anal. Biochem. 162 (1987) 156-159.20 μgtotal RNA was separated on a 1.2% agarose formaldehyde gel andtransferred onto nylon membranes (Amersham, Braunschweig, DE) bystandard methods (Sambrook et al., Molecular Cloning: A laboratorymanual (1989) Cold Spring Harbor Laboratory Press, New York, USA. TheDNA sequence SEQ ID NO:1 or SEQ ID NO:5 was radioactively labeled asprobes (Feinberg, A. P., and Vogelstein, B., Anal. Biochem. 137 (1984)266-267). The hybridization was carried out at 68° C. in 5× SSC, 5×Denhardt, 7% SDS/0.5 M phosphate buffer pH 7.0, 10% dextran sulfate and100 μg/ml salmon sperm DNA. Subsequently the membranes were washed twicefor one hour each time in 1× SSC at 68° C. and then exposed to X-rayfilm.

Example 7

[0127] Procedure for Identification of Modulators of the Activity of theProtein According to the Invention

[0128] The expression vector of Example 5 (either for IIP-1 or IIP-10 10μg/10⁶ cells) is transferred into NIH 3T3 cells by standard methodsknown in the art (Sambrook et al.). Cells which have taken up the vectorare identified by their ability to grow in the presence of the selectionor under selective conditions (0.4 mg/ml G418). Cells which express DNAencoding IIP produce RNA which is detected by Northern blot analysis asdescribed in Example 5. Alternatively, cells expressing the protein areidentified by identification of the protein by Western blot analysisusing the antibodies described in Example 4. Cells which express theprotein from the expression vector will display an altered morphologyand/or enhanced growth properties.

[0129] Cells which express the protein and display one or more of thealtered properties described above are cultured with and without aputative modulator compound. By screening of chemical and naturallibraries, such compounds can be identified using high throughputcellular assays monitoring cell growth (cell proliferation assays usingas chromogenic substrates the tetrazolium salts WST-1, MTT, or XTT, or acell death detection ELISA using bromodesoxyuridine (BrdU); cf.Boehringer Mannheim GmbH, Apoptosis and Cell Proliferation, 2^(nd)edition, 1998, pp. 70-84).

[0130] The modulator compound will cause an increase or a decrease inthe cellular response to the IIP protein activity and will be either anactivator or an inhibitor of IGF-receptor function, respectively.

[0131] Alternatively, putative modulators are added to cultures of tumorcells, and the cells display an altered morphology and/or displayreduced or enhanced growth properties. A putative modulator compound isadded to the cells with and without IIP protein and a cellular responseis measured by direct observation of morphological characteristics ofthe cells and/or the cells are monitored for their growth properties.The modulator compound will cause an increase or a decrease in thecellular response to IIP protein and will be either an activator or aninhibitor of IGF-1 receptor activity, respectively.

[0132] List of References

[0133] Altschul, S. F., et al., J. Mol. Biol. 215 (1990) 403-410

[0134] Altschul, S. F., et al., Nucleic Acids Res. 25 (1997) 3389-3402

[0135] Ausubel I., Frederick M., Current Protocols in Mol Biol. (1992),John Wiley and Sons, New York

[0136] Weidner, K. M., et al., Nature 384 (1996) 173-176); Behrens, J.,et al., Nature 382 (1996) 638-642

[0137] Behrens, J., et al., Science 280 (1998) 596-599

[0138] Boehringer Mannheim GmbH, Apoptosis and Cell Proliferation, 2ndedition, 1998, pp. 70-84

[0139] Büttner et al., Mol. Cell. Biol. 11 (1991) 3573-3583

[0140] Cabral, J. H., et al., Nature 382 (1996) 649-652

[0141] Chomcszynski and Sacchi, Anal. Biochem. 162 (1987) 156-159

[0142] Chowdhury, K., et al., Mech. Dev. 39 (1992) 129-142

[0143] Cooke, M. P., and Perlmutter, R. M., New Biol. 1 (1989) 66-74

[0144] Database EMBL Nos. AF089818 and AF061263

[0145] Denny, P., and Ashworth, A., Gene 106 (1991) 221-227

[0146] DeVries, L., et al., Proc. Natl. Acad. Sci. USA 95 (1998)12340-12345

[0147] Dey, R. B., et al., Mol. Endocrinol. 10 (1996) 631-641

[0148] EP-A 0 063 879

[0149] EP-A 0 128 018

[0150] EP-A 0 173 251

[0151] EP-A 0 200 362

[0152] Feinberg, A. P., and Vogelstein, B., Anal. Biochem. 137 (1984)266-267

[0153] Fields, S., and Song, O., Nature 340 (1989) 245-246

[0154] Goulding, M. D., et al., EMBO J. 10 (1991) 1135-1147

[0155] Hames, B. D., Higgins, S. G., Nucleic acid hybridisation—apractical approach (1985) IRL Press, Oxford, England

[0156] Lehmann, J. M., et al., Nucleic Acids Res. 18 (1990) 1048)

[0157] Margolis, B. L., et al., Proc. Natl. Acad. Sci. USA 89 (1992)8894-8898

[0158] Needleman and Wunsch, J. Biol. Chem. 48 (1970) 443-453

[0159]PCR—A Practical Approach (1991), publ. M. J. McPherson, P. Quirke,G. R. Taylor, IRL Press

[0160] Pearson, W. R., Methods in Enzymology 183 (1990) 63-68, AcademicPress, San Diego, US

[0161] Ponting, C. P., et al., BioEssays 19 (1997) 469-479

[0162]PCR Protocols—A Guide to Methods and Applications (1990), publ. M.A. Innis, D. H. Gelfand, J. J. Sninsky, T. J. White, Academic Press Inc.

[0163] Riedel, H., et al., J. Biochem. 122 (1997) 1105-1113

[0164] Rousset, R., et al., Oncogene 16 (1998) 643-654

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[0168] USP 2915082

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[0170] Weidner, K. M., et al., Nature 384 (1'996) 173-176

[0171] WO 97/27296

[0172] WO 89/06698

[0173] WO 95/14772

[0174] Yokouchi, M., et al., Oncogene 15 (1998) 7-15

1 10 1 1707 DNA Homo sapiens n at position 186, 187, 203, and 205 is a,t, g, or c. 1 gaaacccaca ggaggcaacc acactagttt agatcttctg gtgaccccacttctcgctgc 60 tcatgccgct gggactgggg cggcggaaaa aggcgccccc tctagtggaaaatgaggagg 120 ctgagccagg ccgtggaggg ctgggcgtgg gggagccagg gcctctgggcggaggtgggt 180 cggggnnccc ccaaatgggc ttncnccccc ctcccccagc cctgcggccccgcctcgtgt 240 tccacaccca gctggcccat ggcagtccca ctggccgcat cgagggcttcaccaacgtca 300 aggagctgta tggcaagatc gccgaggcct tccgcctgcc aactgccgaggtgatgttct 360 gcaccctgaa cacccacaaa gtggacatgg acaagctcct ggggggccagatcgggctgg 420 aggacttcat cttcgcccac gtgaaggggc agcgcaagga ggtggaggtgttcaagtcgg 480 aggatgcact cgggctcacc atcacggaca acggggctgg ctacgccttcatcaagcgca 540 tcaaggaggg cagcgtgatc gaccacatcc acctcatcag cgtgggcgacatgatcgagg 600 ccattaacgg gcagagcctg ctgggctgcc ggcactacga ggtggcccggctgctcaagg 660 agctgccccg aggccgtacc ttcacgctga agctcacgga gcctcgcaaggccttcgaca 720 tgatcagcca gcgttcagcg ggtggccgcc ctggctctgg cccacaactgggcactggcc 780 gagggaccct gcggctccga tcccggggcc ccgccacggt ggaggatctgccctctgcct 840 ttgaagagaa ggccattgag aaggtggatg acctgctgga gagttacatgggtatcaggg 900 acacggagct ggcagccacc atggtggagc tgggaaagga caaaaggaacccggatgagc 960 tggccgaggc cctggacgaa cggctgggtg actttgcctt ccctgacgagttcgtctttg 1020 acgtctgggg cgccattggg gacgccaagg tcggccgcta ctaggactgcccccggaccc 1080 tgcgatgatg acccgggcgc aacctggtgg gggcccccag cagggacactgacgtcagga 1140 cccgagcctc cagcctgagc ctagctcagc agcccaagga cgatggtgaggggaggtggg 1200 gccaggcccc ctgccccgct ccactcggta ccatcccctc cctggttcccagtctggccg 1260 gggtccccgg cccccctgtg ccctgttccc cacctacctc agctgggtcaggcacaggga 1320 ggggagggat cagccaaatt gggcggccac ccccgcctcc accactttccaccatcagct 1380 gccaaactgg tccctctgtc tccctggggc cttgggttct gtttgggggtcatgaccttc 1440 ctagtttcct gacgcaggga atacagggga gagggttgtc cttccccccagcaaatgcaa 1500 taatgccctc acccctcctg agaggagccc cctccctgtg gagcctgttacctccgcatt 1560 tgacacgagt ctgctgtgaa ccccgcaacc tcctccccac ctcccatctctccttccagg 1620 cccatccctg gcccagagca ggagggaggg agggacgatg gcggtgggtttttgtatctg 1680 aatttgctgt cttgaacata aagaatc 1707 2 333 PRT Homosapiens Xaa at position 42, 47, and 48 is any one of the twentynaturally occurring amino acids. 2 Met Pro Leu Gly Leu Gly Arg Arg LysLys Ala Pro Pro Leu Val Glu 1 5 10 15 Asn Glu Glu Ala Glu Pro Gly ArgGly Gly Leu Gly Val Gly Glu Pro 20 25 30 Gly Pro Leu Gly Gly Gly Gly SerGly Xaa Pro Gln Met Gly Xaa Xaa 35 40 45 Pro Pro Pro Pro Ala Leu Arg ProArg Leu Val Phe His Thr Gln Leu 50 55 60 Ala His Gly Ser Pro Thr Gly ArgIle Glu Gly Phe Thr Asn Val Lys 65 70 75 80 Glu Leu Tyr Gly Lys Ile AlaGlu Ala Phe Arg Leu Pro Thr Ala Glu 85 90 95 Val Met Phe Cys Thr Leu AsnThr His Lys Val Asp Met Asp Lys Leu 100 105 110 Leu Gly Gly Gln Ile GlyLeu Glu Asp Phe Ile Phe Ala His Val Lys 115 120 125 Gly Gln Arg Lys GluVal Glu Val Phe Lys Ser Glu Asp Ala Leu Gly 130 135 140 Leu Thr Ile ThrAsp Asn Gly Ala Gly Tyr Ala Phe Ile Lys Arg Ile 145 150 155 160 Lys GluGly Ser Val Ile Asp His Ile His Leu Ile Ser Val Gly Asp 165 170 175 MetIle Glu Ala Ile Asn Gly Gln Ser Leu Leu Gly Cys Arg His Tyr 180 185 190Glu Val Ala Arg Leu Leu Lys Glu Leu Pro Arg Gly Arg Thr Phe Thr 195 200205 Leu Lys Leu Thr Glu Pro Arg Lys Ala Phe Asp Met Ile Ser Gln Arg 210215 220 Ser Ala Gly Gly Arg Pro Gly Ser Gly Pro Gln Leu Gly Thr Gly Arg225 230 235 240 Gly Thr Leu Arg Leu Arg Ser Arg Gly Pro Ala Thr Val GluAsp Leu 245 250 255 Pro Ser Ala Phe Glu Glu Lys Ala Ile Glu Lys Val AspAsp Leu Leu 260 265 270 Glu Ser Tyr Met Gly Ile Arg Asp Thr Glu Leu AlaAla Thr Met Val 275 280 285 Glu Leu Gly Lys Asp Lys Arg Asn Pro Asp GluLeu Ala Glu Ala Leu 290 295 300 Asp Glu Arg Leu Gly Asp Phe Ala Phe ProAsp Glu Phe Val Phe Asp 305 310 315 320 Val Trp Gly Ala Ile Gly Asp AlaLys Val Gly Arg Tyr 325 330 3 380 DNA Homo sapiens n at position 369 isa, t, g, or c. 3 gccgaggaag gagaaggggc taaaccttgg agagtggatg gctcaaaggattctcagatc 60 acacctcggg aggatcatgg gcaggagagc ctgttggcag ggctccacggaacgcatcca 120 ccaaagacaa ggcagaaagt cactgcccaa gccggaggcc ccggggatcccatgcttttt 180 tcaagcccag agacagatga gaagcttttt atatgtgcgc agtgtggcaaaaccttcaac 240 aatacctcca acctgagaac gcaccagcgg atccacactg gcgagaagccctacatgtgt 300 tccgagtgtg gcaagagttt ctcccggagc tccaaccgca tccggcacgagcgcatccac 360 ctggaagana agcactctga 380 4 126 PRT Homo sapiens Xaa atposition 123 is any one of the twenty naturally occurring amino acids. 4Ala Glu Glu Gly Glu Gly Ala Lys Pro Trp Arg Val Asp Gly Ser Lys 1 5 1015 Asp Ser Gln Ile Thr Pro Arg Glu Asp His Gly Gln Glu Ser Leu Leu 20 2530 Ala Gly Leu His Gly Thr His Pro Pro Lys Thr Arg Gln Lys Val Thr 35 4045 Ala Gln Ala Gly Gly Pro Gly Asp Pro Met Leu Phe Ser Ser Pro Glu 50 5560 Thr Asp Glu Lys Leu Phe Ile Cys Ala Gln Cys Gly Lys Thr Phe Asn 65 7075 80 Asn Thr Ser Asn Leu Arg Thr His Gln Arg Ile His Thr Gly Glu Lys 8590 95 Pro Tyr Met Cys Ser Glu Cys Gly Lys Ser Phe Ser Arg Ser Ser Asn100 105 110 Arg Ile Arg His Glu Arg Ile His Leu Glu Xaa Lys His Ser 115120 125 5 678 DNA Homo sapiens 5 atgtcgagac cccggaagag gctggctgggacttctggtt cagacaaggg actatcagga 60 aaacgcacca aaactgagaa ctcaggtgaggcattagcta aagtggagga ctccaaccct 120 cagaagactt cagccactaa aaactgtttgaagaatctaa gcagccactg gctgatgaag 180 tcagagccag agagccgcct agagaaaggtgtagatgtga agttcagcat tgaggatctc 240 aaagcacagc ccaaacagac aacatgctgggatggtgttc gtaactacca ggctcggaac 300 ttccttagag ccatgaagct gggagaagaagccttcttct accatagcaa ctgcaaagag 360 ccaggcatcg caggactcat gaagatcgtgaaagaggctt acccagacca cacacagttt 420 gagaaaaaca atccccatta tgacccatctagcaaagagg acaaccctaa gtggtccatg 480 gtggatgtac agtttgttcg gatgatgaaacgtttcattc ccctggctga gctcaaatcc 540 tatcatcaag ctcacaaagc tactggtggccccttaaaaa atatggttct cttcactcgc 600 cagagattat caatccagcc cctgacccaggaagagtttg attttgtttt gagcctggag 660 gaaaaggaac caagttaa 678 6 225 PRTHomo sapiens 6 Met Ser Arg Pro Arg Lys Arg Leu Ala Gly Thr Ser Gly SerAsp Lys 1 5 10 15 Gly Leu Ser Gly Lys Arg Thr Lys Thr Glu Asn Ser GlyGlu Ala Leu 20 25 30 Ala Lys Val Glu Asp Ser Asn Pro Gln Lys Thr Ser AlaThr Lys Asn 35 40 45 Cys Leu Lys Asn Leu Ser Ser His Trp Leu Met Lys SerGlu Pro Glu 50 55 60 Ser Arg Leu Glu Lys Gly Val Asp Val Lys Phe Ser IleGlu Asp Leu 65 70 75 80 Lys Ala Gln Pro Lys Gln Thr Thr Cys Trp Asp GlyVal Arg Asn Tyr 85 90 95 Gln Ala Arg Asn Phe Leu Arg Ala Met Lys Leu GlyGlu Glu Ala Phe 100 105 110 Phe Tyr His Ser Asn Cys Lys Glu Pro Gly IleAla Gly Leu Met Lys 115 120 125 Ile Val Lys Glu Ala Tyr Pro Asp His ThrGln Phe Glu Lys Asn Asn 130 135 140 Pro His Tyr Asp Pro Ser Ser Lys GluAsp Asn Pro Lys Trp Ser Met 145 150 155 160 Val Asp Val Gln Phe Val ArgMet Met Lys Arg Phe Ile Pro Leu Ala 165 170 175 Glu Leu Lys Ser Tyr HisGln Ala His Lys Ala Thr Gly Gly Pro Leu 180 185 190 Lys Asn Met Val LeuPhe Thr Arg Gln Arg Leu Ser Ile Gln Pro Leu 195 200 205 Thr Gln Glu GluPhe Asp Phe Val Leu Ser Leu Glu Glu Lys Glu Pro 210 215 220 Ser 225 7 18DNA Artificial Sequence Description of Artificial Sequenceprimer TIP2c-s7 gaaacccaca ggaggcaa 18 8 18 DNA Artificial Sequence Description ofArtificial Sequenceprimer TIP2b-r 8 ggtcatcatc gcagggtc 18 9 33 DNAArtificial Sequence Description of Artificial Sequenceprimer Hcthy-s 9agcttgcggc cgcagatgtc gagaccccgg aag 33 10 40 DNA Artificial SequenceDescription of Artificial Sequenceprimer Hcthy-r 10 agcttgcggccgcgaattct taacttggtt ccttttcctc 40

What is claimed is:
 1. A recombinant polypeptide which binds to theIGF-1 receptor, wherein the recombinant polypeptide is encoded by anucleic acid selected from the group consisting of: a) the nucleic acidsshown in SEQ ID NO:5 or a nucleic acid sequence which is complementarythereto; b) nucleic acids which hybridize under stringent conditionswith one of the nucleic acids from a) encoding a polypeptide showinghomology with the polypeptide of SEQ ID NO:6; and c) sequences that dueto the degeneracy of the genetic code encode IIP-10 polypeptides havingthe amino acid sequence of the polypeptides encoded by the sequences ofa) and b).
 2. A recombinant polypeptide according to claim 1, whereinthe hybridization in b) is performed in 5.0× SSC, 5× Denhardt, 7% SDS,0.5 M phosphate buffer pH 7.0, 10% dextran sulfate and 100 μg/ml salmonsperm DNA at about 50° C.-68° C., followed by two washing steps with 1×SSC at 68° C.
 3. A method for the detection of the proliferationpotential of a cancer cell comprising a) incubating a sample wherebysaid sample contains nucleic acids with a nucleic acid probe which isselected from the group consisting of: (i) the nucleic acid shown in SEQID NOS:1, 3 or 5 or a nucleic acid which is complementary thereto; and(ii) nucleic acids which hybridize with one of the nucleic acids from(i) and b) detecting the hybridization by means of a further bindingpartner of the nucleic acid of the sample and/or the nucleic acid probe.4. The method of claim 3 wherein said sample is selected from the groupconsisting of body fluid of a patient suffering from cancer; tumorcells; a tumor cell extract; and a cell culture supernatant of saidtumor cells.
 5. The method of claim 3, wherein hybridization is effectedat least with the nucleic acid fragment of SEQ ID NO:1 or SEQ ID NO:5 orthe complementary fragment.
 6. The method of claim 3 wherein the nucleicacid to be detected is amplified before the detection.
 7. The method ofclaim 5 wherein the nucleic acid to be detected is amplified before thedetection.
 8. A method for screening a compound that inhibits theinteraction between IGF-1R and IIP-10 comprising: a) combining IGF-1Rand said IIP polypeptide with a solution containing a candidate compoundsuch that the IGF-1R and said IIP polypeptide are capable of forming acomplex and b) determining the amount of complex relative to thepredetermined level of binding in the absence of the compound andtherefrom evaluating the ability of the compound to inhibit binding ofIGF-1R to said IIP.
 9. A method for the production of a therapeuticagent for the treatment of carcinomas in a patient comprising combininga pharmaceutically acceptable carrier with a therapeutically effectiveamount of a compound which modulates the interaction between IGF-1R andIIP-10 in a cellular assay, whereby in said cellular assay tumor cellsor cells transfected with expression constructs of IGF-1R and of saidIIP are treated with said compound, and complex formation between IGF-1Rand said respective IIP is analyzed, and the extent of said complexformation in the case of inhibition does not exceed 50% when referencedagainst100% for complex formation without said compound in said samecellular assay.
 10. A method according to claim 9, wherein the compoundinhibits the interaction.